Kidney functional reserve and damage biomarkers in subclinical chronic kidney disease and acute kidney injury.

Significant loss of kidney function is not easily identified by serum creatinine (sCr)-based measurements. In the presence of normal sCr, decreased kidney functional reserve (KFR) may identify a significant loss of function. We evaluated KFR in experimental subclinical chronic kidney disease (sCKD) before and after brief ischemia-reperfusion injury (IRI). Using fluorescein isothiocyanate-labeled sinistrin, glomerular filtration rate (GFR) was measured transcutaneously before and after adenine-induced sCKD, and 1 and 2 wk after brief IRI, and compared with urinary kidney damage biomarkers. sCKD reduced stimulated and unstimulated GFR by ∼20% while reducing KFR by 50%. IRI reduced unstimulated GFR for 14 days, but KFR remained relatively unchanged in sCKD and transiently increased in control kidneys at 7 days. sCr increased and creatinine clearance (CrCl) decreased only immediately after IRI; sCr and CrCl correlated poorly with measured GFR except on day 1 after IRI. Heterogeneity in sCr and CrCl resulted from variation in tubular creatinine secretion. The increase in damage biomarker concentrations persisted for up to 14 days after IRI, allowing retrospective detection of sCKD before AKI by urine clusterin/urine kidney injury molecule-1 with an area under the curve of 1.0. sCr and CrCl are unreliable unless sCr is acutely elevated. Measurement of KFR and urine damage biomarker excretion detected sCKD despite normal sCr and CrCl. After IRI, the urine clusterin-to-urine kidney injury molecule-1 ratio may identify prior sCKD.NEW & NOTEWORTHY Early kidney function loss is poorly identified by serum creatinine (sCr)-based measurements. Direct kidney functional reserve (KFR) measurement before kidney injury and elevated urinary biomarkers clusterin and kidney injury molecule-1 detect subclinical chronic kidney disease (sCKD) after kidney injury despite normal range sCr and creatinine clearance. Reliance on sCr masks underlying sCKD. Acute kidney injury risk evaluation requires direct glomerular filtration rate measurement and KFR, whereas kidney damage biomarkers facilitate identification of prior subclinical injury.

Acetylated histone variant H2A.Z is involved in the activation of neo-enhancers in prostate cancer.

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active promoters and is associated with oncogene activation in prostate cancer, but its role in enhancer function is still poorly understood. Here we show that H2A.Zac containing nucleosomes are commonly redistributed to neo-enhancers in cancer resulting in a concomitant gain of chromatin accessibility and ectopic gene expression. Notably incorporation of acetylated H2A.Z nucleosomes is a pre-requisite for activation of Androgen receptor (AR) associated enhancers. H2A.Zac nucleosome occupancy is rapidly remodeled to flank the AR sites to initiate the formation of nucleosome-free regions and the production of AR-enhancer RNAs upon androgen treatment. Remarkably higher levels of global H2A.Zac correlate with poorer prognosis. Altogether these data demonstrate the novel contribution of H2A.Zac in activation of newly formed enhancers in prostate cancer.

SUMOylation of GTF2IRD1 regulates protein partner interactions and ubiquitin-mediated degradation.

GTF2IRD1 is one of the genes implicated in Williams-Beuren syndrome, a disease caused by haploinsufficiency of certain dosage-sensitive genes within a hemizygous microdeletion of chromosome 7. GTF2IRD1 is a prime candidate for some of the major features of the disease, presumably caused by abnormally reduced abundance of this putative transcriptional repressor protein. GTF2IRD1 has been shown to interact with the E3 SUMO ligase PIASxß, but the significance of this relationship is largely unexplored. Here, we demonstrate that GTF2IRD1 can be SUMOylated by the SUMO E2 ligase UBC9 and the level of SUMOylation is enhanced by PIASxß. A major SUMOylation site was mapped to lysine 495 within a conserved SUMO consensus motif. SUMOylation of GTF2IRD1 alters the affinity of the protein for binding partners that contain SUMO-interacting motifs, including a novel family member of the HDAC repressor complex, ZMYM5, and PIASxß itself. In addition, we show that GTF2IRD1 is targeted for ubiquitination and proteasomal degradation. Cross regulation by SUMOylation modulates this process, thus potentially regulating the level of GTF2IRD1 protein in the cell. These findings, concerning post-translational control over the activity and stability of GTF2IRD1, together with previous work showing how GTF2IRD1 directly regulates its own transcription levels suggest an evolutionary requirement for fine control over GTF2IRD1 activity in the cell.

GTF2IRD2 from the Williams-Beuren critical region encodes a mobile-element-derived fusion protein that antagonizes the action of its related family members.

GTF2IRD2 belongs to a family of transcriptional regulators (including TFII-I and GTF2IRD1) that are responsible for many of the key features of Williams-Beuren syndrome (WBS). Sequence evidence suggests that GTF2IRD2 arose in eutherian mammals by duplication and divergence from the gene encoding TFII-I. However, in GTF2IRD2, most of the C-terminal domain has been lost and replaced by the domesticated remnant of an in-frame hAT-transposon mobile element. In this first experimental analysis of function, we show that transgenic expression of each of the three family members in skeletal muscle causes significant fiber type shifts, but the GTF2IRD2 protein causes an extreme shift in the opposite direction to the two other family members. Mating of GTF2IRD1 and GTF2IRD2 mice restores the fiber type balance, indicating an antagonistic relationship between these two paralogs. In cells, GTF2IRD2 localizes to cytoplasmic microtubules and discrete speckles in the nuclear periphery. We show that it can interact directly with TFII-Iß and GTF2IRD1, and upon co-transfection changes the normal distribution of these two proteins into a punctate nuclear pattern typical of GTF2IRD2. These data suggest that GTF2IRD2 has evolved as a regulator of GTF2IRD1 and TFII-I; inhibiting their function by direct interaction and sequestration into inactive nuclear zones.

Mutation of Gtf2ird1 from the Williams-Beuren syndrome critical region results in facial dysplasia, motor dysfunction, and altered vocalisations.

Insufficiency of the transcriptional regulator GTF2IRD1 has become a strong potential explanation for some of the major characteristic features of the neurodevelopmental disorder Williams-Beuren syndrome (WBS). Genotype/phenotype correlations in humans indicate that the hemizygous loss of the GTF2IRD1 gene and an adjacent paralogue, GTF2I, play crucial roles in the neurocognitive and craniofacial aspects of the disease. In order to explore this genetic relationship in greater detail, we have generated a targeted Gtf2ird1 mutation in mice that blocks normal GTF2IRD1 protein production. Detailed analyses of homozygous null Gtf2ird1 mice have revealed a series of phenotypes that share some intriguing parallels with WBS. These include reduced body weight, a facial deformity resulting from localised epidermal hyperplasia, a motor coordination deficit, alterations in exploratory activity and, in response to specific stress-inducing stimuli; a novel audible vocalisation and increased serum corticosterone. Analysis of Gtf2ird1 expression patterns in the brain using a knock-in LacZ reporter and c-fos activity mapping illustrates the regions where these neurological abnormalities may originate. These data provide new mechanistic insight into the clinical genetic findings in WBS patients and indicate that insufficiency of GTF2IRD1 protein contributes to abnormalities of facial development, motor function and specific behavioural disorders that accompany this disease.

Negative autoregulation of GTF2IRD1 in Williams-Beuren syndrome via a novel DNA binding mechanism.

The GTF2IRD1 gene is of principal interest to the study of Williams-Beuren syndrome (WBS). This neurodevelopmental disorder results from the hemizygous deletion of a region of chromosome 7q11.23 containing 28 genes including GTF2IRD1. WBS is thought to be caused by haploinsufficiency of certain dosage-sensitive genes within the deleted region, and the feature of supravalvular aortic stenosis (SVAS) has been attributed to reduced elastin caused by deletion of ELN. Human genetic mapping data have implicated two related genes GTF2IRD1 and GTF2I in the cause of some the key features of WBS, including craniofacial dysmorphology, hypersociability, and visuospatial deficits. Mice with mutations of the Gtf2ird1 allele show evidence of craniofacial abnormalities and behavioral changes. Here we show the existence of a negative autoregulatory mechanism that controls the level of GTF2IRD1 transcription via direct binding of the GTF2IRD1 protein to a highly conserved region of the GTF2IRD1 promoter containing an array of three binding sites. The affinity for this protein-DNA interaction is critically dependent upon multiple interactions between separate domains of the protein and at least two of the DNA binding sites. This autoregulatory mechanism leads to dosage compensation of GTF2IRD1 transcription in WBS patients. The GTF2IRD1 promoter represents the first established in vivo gene target of the GTF2IRD1 protein, and we use it to model its DNA interaction capabilities.